GENE MODIFICATION

Question 1

Totipotent Cells

Answer 1

cells that can mature into any type of body cell, i.e. they are undifferentiated

Question 2

Where we get totipotent cells from

Answer 2

adult stem cells, embryonic stem cells (however they only occur for a limited amount of time), induced pluripotent stem cells
When a cell becomes specialised, we say it has lost its totipotency. Whilst it has totipotency, it can be used to treat genetic diseases.

Question 3

Features of totipotent cells

Answer 3

Will replace themselves, i.e. they are immortal (except for multipotent cells which divide to form only a limited number)
They are undifferentiated

Question 4

In plants some cells remain totipotent throughout life. These plants are used naturally and artificially in 3 ways

Answer 4

Vegetative Propagation
Cuttings
Tissue culture

Question 5

Cells specialise themselves by controlling which genes are expressed in two ways

Answer 5

Oestrogen; only works in Target Cells, target cells are cells
SiRNA

Question 6

Oestrogen

Answer 6

Transcription begins when the gene being transcribed is stimulated by Transcription Factors
Transcription Factors have an active site which binds to a specific sequence of DNA to start transcription, this specific sequence is called the PROMOTER SEQUENCE
This active site is blocked by an inhibitor molecule which is attached to the Transcription Factor and Receptor, this inhibitor molecule prevents transcription and thus prevents the gene from being expressed
Oestrogen diffuses into cell via LIPID DIFFUSION
Oestrogen then binds to the active site of the RECEPTOR
This changes the shape of the RECEPTOR molecules, therefore releasing the inhibitor molecule and thus leaving the active site of the Transcription Factor vacant
This allows Transcription Factor to ENTER THE NUCLEUS and attach itself to a specific base sequence on a DNA promoter

Question 7

How Transcriptional Factors are important for synthesis of particular proteins

Answer 7

They bind to DNA at specific region called the PROMOTER SEQUENCE
They then stimulate RNA Polymerase to arrive and begin transcription

If we want a cell to stop expressing its genes, we can introduce a molecule to compete with Oestrogen for the active site of the RECEPTOR.

Question 8

SiRNA

Answer 8

An enzyme cuts large double stranded RNA into smaller sections called Small Interfering RNA (SiRNA)
One of the strands of the SiRNA combines with an enzyme called RNA-induced silencing complex
SiRNA guides enzyme to mRNA and forms complimentary base pairs with a specific mRNA
The enzyme still attached to the SiRNA then breaks the mRNA into smaller pieces
mRNA no longer capable of translation
Therefore target gene can no longer make a specific protein as mRNA has been cut into pieces
SiRNA is used to block genes that cause diseases, i.e. can be used to stop genetic diseases by stopping the gene from producing proteins.

Question 9

Genetic Modification

Answer 9

Isolation: finding DNA fragments that have the desired gene that produces the desired protein, then separating it
Insertion: putting the desired DNA fragments into a vector
Transformation: transferring the DNA into a suitable host cell
Identification: deducing which host cells have taken up the DNA and which haven’t
Growth: making more copies of the host cell

Question 10

Isolation can be done in two ways

Answer 10

Use Reverse Transcriptase
Reverse Transcriptase catalyses the production of DNA FROM RNA
PROCESS:
Find cell that produces desired protein and remove mRNA from this cell
Add Reverse Transcriptase to make a strand of DNA from mRNA, this strand that is made is called Complementary DNA (cDNA)
To make the other strand of DNA, the enzyme DNA Polymerase is used to form complimentary base pairs with cDNA, therefore cDNA acts as a template for the second strand

Question 11

Using Reverse Transcriptase is better than taking actual DNA from the organism because

Answer 11

DNA contains Introns too whereas mRNA will contain only activated desired genes
Makes stable copy of a gene since DNA is less readily broken down by enzymes than RNA
It makes genes easier to find, as there are thousands of genes but only a few types of mRNA exist

Question 12

Isolation: Use Restriction Endonucleases

Answer 12

Restriction Endonucleases cut a DNA double strand VIA HYDROLYSIS at a specific sequence of bases called a recognition sequence in order to isolate the useful DNA sequence
Usually, the DNA sequence the Restriction Endonucleases will cut will be a PALINDROMIC SEQUENCE, which means: sequence of one strand is the reversal of the corresponding strand.

Question 13

Restriction Endonucleases cuts DNA in two ways

Answer 13

Blunt Ends and Sticky Ends
The sticky ends/blunt ends can be attached to other sticky ends/blunt ends that have been cut by the SAME Restriction Endonucleases using DNA LIGASE to form a Recombinant DNA VIA CONDENSATION:

Sometimes, there are quite a few recognition sequences leading to Restriction Endonucleases causing many fragments, but scientists only need one specific fragment containing the desired DNA fragment, therefore they will use Gel Electrophoresis to separate the fragments. (Gel Electrophoresis will be explained shortly) Once the fragments are separated, they will use a DNA Probe (will be explained shortly) which will bind to a complimentary base sequence in the gene, the fluorescence/radioactivity of the DNA Probe will tell us which fragment contains the desired gene. Then we will movie onto insertion, etc.

Question 14

The overall isolation using Restriction Endonucleases

Answer 14

Restriction enzymes cut DNA at specific base sequences VIA HYDROLYSIS forming a sticky end
Same restriction enzyme also cuts DNA into which gene is inserted forming another sticky end
DNA Ligase joins the two pieces of DNA together which happen to be complimentary VIA CONDENSATION to form recombinant DNA

Question 15

Why DNA base sequences must be cut with the SAME Restriction Endonucleases

Answer 15

So that it cuts VIA HYDROLYSIS at the same base sequence therefore allowing pairing of bases when DNA Ligase is used

Question 16

Why Restriction Endonucleases (enzyme) will cut DNA only at specific recognition sites

Answer 16

Different lengths of DNA have different base sequences
The enzyme’s active site has different shapes
Therefore only specific sequence will fit active site of an enzyme

Question 17

One the DNA sequence has been isolated; we may continue the process of insertion, transformation, etc. in two ways

Answer 17

In Vivo: using living cells

In Vitro: using the Polymerase Chain Reaction

Question 18

Before a plasmid with the desired gene is put into, the bacteria, the gene for conjugation is removed from the plasmid because

Answer 18

This prevents bacteria cells from conjugating

Therefore stops the transfer of DNA, thus reducing the risk of other organisms getting altered genes

Question 19

Why biologists will often use plasmids which contain antibiotic resistant genes

Answer 19

It can act as a marker

This allows detection of the cells containing the desired DNA

Question 20

If in the exam it asks for the definition of the term ‘sticky ends’, you will write

Answer 20

Cut ends of DNA
One strand longer than the other
Can attach to complimentary DNA

Question 21

In Vivo

Answer 21

1) Isolation: This has been completed
2) Insertion: putting the desired DNA fragments into a vector
Using Restriction Endonucleases we can combine the DNA of one organism with that of another organism as long as the Restriction Endonucleases used to create the sticky ends in both was the same.

Question 22

PROCESS OF INSERTION

Answer 22

DNA isolated from cell which manufactures desired protein
DNA and a plasmid both cut using the same Restriction Endonucleases, therefore creating DNA fragment with sticky end and plasmid with a hole that has a sticky end
DNA fragments and plasmid with holes are mixed together with DNA LIGASE, the DNA fragment fits into the hole in the plasmid like a jigsaw puzzle therefore forming recombinant DNA
The plasmids containing the DNA fragment will be the vectors, which are materials used to transport DNA into the HOST CELL

Question 23

Recombinant DNA

Answer 23

Contains genes of 2 types of organisms (plasmid will become like this)

Question 24

If asked in the exam, how a DNA fragment in inserted into a vector, you will write

Answer 24

Cut vector DNA with same Restriction Endonucleases used to cut DNA containing desired gene
Use DNA Ligase to join the sticky ends

Question 25

Why plasmid is described as a vector

Answer 25

It can carry foreign DNA into a bacteria cell

Question 26

Properties of a vector

Answer 26

Big enough to hold gene Circular so that it is less likely to be broken down Contains control sequences so that it can replicate the gene

Question 27

After the DNA fragment is inserted into the plasmid, the plasmid will function differently, i.e. if you replaced a resistance gene with the desired gene, then the plasmid will no longer be resistant like it was before because:

Answer 27

The plasmid DNA base sequence has been altered Therefore different proteins will be made Transformation: putting the vector into the host cell Transformation is obtained by mixing the plasmids and bacterial cells together in a medium of Ca2+ ions which causes the bacterial cells to become permeable to the plasmids Identification: deducing which host cells (bacterial cells) have taken up the DNA and which haven’t The method used for Identification is Gene Markers

Question 28

Antibiotic-Resistance Markers

Answer 28

Remove resistance gene to a certain antibiotic from a plasmid and add the desired gene in its place, put this plasmid into bacterial cells Put bacterial cells on nutrient agar plates Transfer a small colony of the bacterial cells onto a second plate in exactly the same position as the original plate Add the antibiotic to the second plate (the antibiotic for which the plasmid containing the desired gene no longer has resistance) The cells that die are the ones that have taken up the desired gene as they have replaced their antibiotic resistance gene with the desired gene The colonies in exactly the same position on the original plate are the ones that possess the required gene

Question 29

Fluorescent Markers

Answer 29

Transfer gene of Jellyfish that produces a green light into the plasmid, put all plasmids into bacterial cells which will all begin to glow Now transplant the desired gene into centre of the green light gene in plasmid The bacterial cells which contain the plasmids that have taken up the desired gene will stop glowing as the desired gene has stopped the green light gene from working

Question 30

Enzyme Markers

Answer 30

Lactase turn colourless substrate into blue Therefore put the desired gene into the gene that makes lactase and insert into plasmid, then insert plasmid into bacterial cells Therefore whichever bacterial cells have a plasmid with the desired gene, their lactase gene will not work Therefore when the bacterial cells are put in a colourless substrate, the bacterial cells containing the plasmid with the required gene will not change to blue

Question 31

Growth

Answer 31

The bacterial cells that have the plasmid with the desired gene are now replicated PROCESS: Using a fermenter

Question 32

Importance of Markers

Answer 32

Allows transformed bacteria to be separate from non-transformed bacteria

Question 33

OVERALL, THE PROCESS OF GENETIC ENGINEERING/GENETIC MODIFICATION

Answer 33

Cut desired gene out of cell using Reverse Transcriptase/Restriction Endonucleases Cut plasmid using the same Restriction Endonucleases Insert the gene into the plasmid, thereby joining the sticky ends using DNA Ligase Transfer the plasmid into a cell Use gene markers to identify which cells have taken up the new gene Cells which have taken up the new gene cloned Sometimes, instead of a bacteria cell, a virus is used for genetic engineering.

Question 34

Advantages of using a virus to introduce genes into cells

Answer 34

Virus can enter cells and replicate inside cells | Viruses target specific cells

Question 35

Disadvantages of using a virus to introduce genes into cells

Answer 35

May cause disease | Immunity may develop to the virus

Question 36

DNA polymerase

Answer 36

An enzyme that joins free nucleotides with their complimentary bases on the DNA, but it does work for the start and end sequence of the DNA strand

Question 37

Primers

Answer 37

have base sequence complimentary to that of the start and end sequence of the DNA to be copied

Question 38

PROCESS (POLYMERASE CHAIN REACTION)

Answer 38

The desired DNA fragments, primers and DNA polymerase are placed in a vessel in a thermocycler The temperature is increased to 95oC, causing the two strands of the desired DNA fragments to separate Mixture is cooled to 55oC, causing primers to join with complimentary bases on the start and end of the two desired DNA strands, therefore original DNA strands are unable to rejoin Now DNA Polymerase begins to join free nucleotides to the middle sections of the two desired DNA strands The temperature is increased to 72oC, this is the optimum temperature for DNA polymerase to work We then have two copies of the original DNA fragment, so the cycle is repeated

Question 39

In the exam, you describe PCR as

Answer 39

Heat DNA to 95oC to allow strands to separate Add primers and nucleotides Cool so that primers bind to DNA and nucleotides attach by complementary base pairing Temperature then taken to 72oC to allow DNA Polymerase to form a new strand

Question 40

How a Polymerase Chain Reaction may stop by itself

Answer 40

Free nucleotides get used up therefore nothing to make complementary chains Primers get used up there cannot start complementary chains Enzymes lose activity so no polymerisation of complementary strands

Question 41

As a recap, the reasons for the following are

Answer 41

Heating sample of DNA above 90oC: separate stranded DNA Adding primers: attaches to start of gene and replication of base sequence starts from here DNA Polymerase used at high temperatures: DNA polymerase not denatured by high temperatures thus allowing rapid replication of DNA

Question 42

How PCR differs from DNA Replication

Answer 42

PCR uses heat to separate strands PCR replicates pieces of DNA because DNA has been cut Primer is added in PCR to initiate replication

Question 43

How PCR differs from transcription

Answer 43

Transcription uses RNA Polymerase, PCR uses DNA Polymerase | In transcription, the template is one strand, in PCR, the template is two strands

Question 44

Advantages of In Vivo

Answer 44

1) Useful when we wish to introduce a gene into another organism 2) No risk of contamination 3) Very accurate 4) Cuts out specific genes 5) transformed bacteria can be used to produce large quantities of gene products

Question 45

Advantages of In Vitro

Answer 45

1) Extremely rapid 2) Does not require living cells 3) Very little purification of final sample needed 4) Very sensitive, can clone DNA molecule up to 1kpb long

Question 46

Disadvantages of In Vivo

Answer 46

1) Relatively slow | 2) complex purification

Question 47

Disadvantages of In Vitro

Answer 47

1) Mistakes in copying in bases take place

Question 48

Cystic Fibrosis

Answer 48

caused by a mutation in the gene for the protein CFTR, which is a chloride ion channel Effect of Cystic Fibrosis: too much sticky mucus produced by epithelial cells

Question 49

Gene Therapy

Answer 49

replacing defective genes with healthy genes Gene Replacement: defective gene replaced with healthy gene Gene Supplementation: healthy copy of gene added alongside defective gene, the healthy genes will be dominant

Question 50

There are two ways in gene therapy may be adopted

Answer 50

Germ-line Therapy: replacing or supplementing the defective gene in the fertilised egg; prohibited due to ethics Somatic-cell Therapy: targets just the affected areas, but the treatment needs to be repeated periodically

Question 51

Somatic-cell Therapy is carried out in two ways

Answer 51

Using a virus called Adenoviruses as vectors for the healthy gene Wrapping the plasmids containing the healthy gene in lipid molecules to form a liposome which can pass through phospholipid bilayer

Question 52

Somatic-cell Therapy is not always effective because:

Answer 52

Adenoviruses may cause infection Patients may develop immunity from Adenoviruses Liposome may not be able to pass through phospholipid bilayer Healthy gene may not be expressed Healthy gene may enter genome of another gene such as tumour suppressor genes, thus causing cancer

Question 53

How a person may become cured through Gene Therapy

Answer 53

Healthy gene is expressed | Healthy genes replicated with cells

Question 54

Why Gene Therapy may still result in children with the genetic disease

Answer 54

Gamete cells do not take up the healthy gene | Therefore the person is able to pass on defective gene

Question 55

Effectiveness of gene therapy

Answer 55

Effect is short lived It can induce an immune response Using viral vectors to deliver the gene present problems The genes are not always expressed Not effective in treating conditions that arise in more than one gene

Question 56

Gene Therapy can also be used to replace the defective gene in a gamete, however this is illegal in the UK because:

Answer 56

Changes to genetic make-up of individual may affect normal development

Question 57

Concerns regarding Gene Therapy

Answer 57

High cost compared with conventional treatments Unknown side-effects Use of animals in preliminary testing Other genes introduced which may have damaging effects